NUCLEIC ACID MODIFICATION WITH SPECIFIC ENDONUCLEASES

用特定核酸内切酶进行核酸修饰

• 批准号: 3270734
• 负责人: HORACE B GRAY
• 金额: $ 10.43万
• 依托单位: UNIVERSITY OF HOUSTON
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-04-01 至 1988-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3270734
• 关键词: DNA; RNA; acidity /alkalinity; affinity chromatography; bacteria; benzopyrenes; chemical cleavage; circular DNA; divalent cations; endonuclease; enzyme structure; enzyme substrate; hydrolysis; molecular cloning; molecular site; nucleic acid structure; nucleobase; pancreatic ribonuclease; phosphomonoesterases; physical separation; radiotracer; temperature

The long-term objectives are to characterize useful nucleases and other extracellular enzymes from the marine bacterial genus Alteromonas. The health relatedness of the project lies in the use of these enzymes in genetic engineering-related manipulations of nucleic acids, for which the nucleases have already proven useful in connection with the cloning and expression in E. coli of such medically significant proteins as insulins and interferons. The nucleases, which exist in at least two molecularly and kinetically distinct forms and possess activities against single-stranded DNA, covalent lesions and other distortions in duplex DNA and the termini of linear duplex DNA, are to be further characterized with respect to the dependence of catalytic behavior on a number of environmental variables. Kinetic and reaction mechanisms studies to be done include characterization of the intermediate and final products of digestion of DNA, the elucidation of the directionality and processivity of the activities on linear duplex DNA and single stranded DNAs, the effects of 2',3'-dideoxy terminal nucleotides and of nucleotide analogs bearing a sulfur atom on the Alpha-phosphate on the exonuclease activity when such groups are present at the termini, measurement of the kinetics of conversion of nicked circular to linear duplex DNA and the determination of the location of the site of cleavage in DNA containing a pre-existing strand break to the site of the initial break. A model for the known generation of ligatable fragments bearing single-stranded termini after partial degradation of linear duplex DNAs will be tested, which is expected to lead to a general method for the detection of palindromic sequences in duplex DNAs. The kinetics of cleavage in nonsupercoiled closed circular DNA reacted with a metabolite of the carcinogen benzo[a]pyrene will be determined. One species of the nuclease can apparently be derived from the other by proteolysis and this is to be confirmed and observed intermediates examined. Determination of whether the nuclease cleaves at potential cruciform structures in supercoiled DNA for which this has been shown to be the case for other single-strand-specific nucleases will be done. Attempts to clone the gene(s) for the nuclease will be made, using as first approaches simple methods developed in this laboratory to assay for periplasmic or extracellular nucleases. Preliminary characterization of a phosphatase in culture supernatants that has 5'-nucleotidase activity will be undertaken.

长期目标是确定有用的核酸酶和其他 海洋细菌属Alteromonas的胞外酶。这个 该项目与健康的相关性在于这些酶在 与基因工程相关的核酸操作，对于这些操作， 核酸酶已被证明与克隆和 胰岛素等具有医学意义的蛋白质在大肠杆菌中的表达 和干扰素。存在于至少两个分子中的核酸酶 和运动上截然不同的形式，并拥有针对 双链DNA中的单链DNA、共价损伤和其他扭曲 和线性双链DNA的末端，将进一步用 关于催化行为对一些 环境变量。动力学和反应机理研究有待于 完成包括对中间产品和最终产品的表征 DNA的消化，阐明DNA的方向性和加工性 对线性双链DNA和单链DNA的活性及其影响 2‘，3’-双脱氧末端核苷酸和含有 硫原子对α-磷酸核酸外切酶活性的影响 基团出现在终端，测量动力学 镍环向线型双链DNA的转化及其含量的测定 含有预先存在的DNA的切割位点的位置 链断裂到最初断裂的位置。已知的典范 含单链末端的可连接片段的产生 将测试线性双链DNA的部分降解，这是预期的 为了得到一种检测回文序列的通用方法， 双链DNA。非超螺旋闭合圆的解理动力学 DNA与致癌物苯并[a]芘的代谢物反应将是 下定决心。一种核酸酶显然可以从 另一种是通过蛋白质分解，这是需要确认和观察的中间产物。 检查过了。核酸酶是否在电位下裂解的测定 超螺旋DNA中的十字形结构，已被证明是 其他单链特异性核酸酶的情况也会这样做。尝试 为了克隆核酸酶基因(S)，将首先使用AS 本实验室开发的简单方法用于检测 胞外或胞外核酸酶。甲藻的初步表征 具有5‘-核苷酸酶活性的培养上清液中的磷酸酶将 要承担的责任。